Effect Of Deletion Of PagP On Gene Expression And Outer Membrane Characteristics Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is a pathogen that causes poultry's local or systemic E.coli disease.At present,it has almost reached the provinces and cities that have a poultry industry in the country,seriously affecting the development of poultry farming,causing serious economic losses.The poultry E.coli disease caused by different serotypes of APEC has seriously jeopardized the healthy development of the world poultry industry and is one of the important bacterial infectious diseases.APEC virulence factors include pilus,toxins,invading enzymes and outer membrane proteins,etc.The pathogenicity is complex and the serotype is diverse.There are many ways to regulate gene transcription by bacteria,and the PhoP/PhoQ binary regulatory system is one of the key pathways.Among them,the regulation of the expression of lipid A related to LPS synthesis is one of its functions.The transcriptional expression of the pagP gene is controlled by the PhoP/PhoQ regulon.Previous studies in this laboratory found that pagP gene expression was significantly up-regulated after the pathogenic E.coli PhoP/PhoQ was knocked out.Hittle et al.reported that pagP of B.parapertussis is involved in the modification of lipid A to regulate bacterial outer membrane function.The gene encoding palmitoyl transferase,pagP,can regulate the outer membrane function of the bacteria by palmitoylating the structure of lipid A of LPS.This palmitoylation of lipid A is closely related to the pathogenicity of bacteria.A modification of the interaction of bacteria with their host during infection.This study selected APEC pagP gene-deleted strains as the research object,and used RNA-Seq technology to sequence and analyze the differentially expressed genes to study the effect of pagP gene deletion on bacterial virulence and its outer membrane characteristics,In order to further study the pathogenic role of pagP gene in avian pathogenic E.coli,it provides a theoretical basis.The main research content is as follows: 1.Effect of avian pathogenic E.coli pagP deletion on virulenceIn this experiment,serum bactericidal and macrophage phagocytosis experiments were used to study the effect of the deletion of the pagP gene on the virulence of APEC.The serum bactericidal test results showed that the deletion of pagP gene can significantly enhance the antiserum of APEC antisera at serum concentrations of 0.5% and 5%(P<0.01).The macrophage phagocytosis experiments showed that the phagocytosis of chicken macrophage with the pagP deleted strain was significantly enhanced compared to the original strain AE17(P<0.05).2.Screening differentially expressed genes of avian pathogenic E.coli pagP deletions based on RNA-SeqIn order to study the effect of the deletion of pagP gene on APEC gene level,the transcriptome technique was used to screen the differentially expressed genes resulting from the deletion of the pagP gene of avian pathogenic E.coli.The results showed 372 differentially expressed genes,down-regulated 33 genes and up-regulated 339 genes.The GO functional annotation shows that the differential genes mainly involved in biological regulation,cellular processes,metabolic processes,responses to stimuli,and other biological processes;the molecular functions involved mainly include catalytic activity,transporter activity,activity of nucleic acid binding transcription factor,protein binding and other functions;the cell components involved mainly include membranes,membrane fractions,cells,and macromolecular complexes.The analysis of KEGG pathway showed that the major enriched pathways of differential genes are pathways such as amino acid metabolism,energy metabolism,lipid metabolism,cell movement,membrane transport,and signal transduction.Genes related to outer membrane proteins and resistance genes such as kpsM,kpsT,ompF,ompW,fyuA,flhD,flhC,evgS,evgA,and acrR were screened out.The expression of some differential genes(ompF,kpsM,fyuA,flhD,yfhD,evgS)was verified by qPCR.The results showed that RNA-Seq data and fluorescence quantitative PCR data were basically consistent,indicating that RNA-Seq results were reliable.3.Effect of avian pathogenic E.coli pagP deletion on bacterial outer membraneIn this experiment,the minimal inhibitory concentration(MIC)experiment was used to explore the effect of the loss of pagP gene on the permeability of the outer membrane of the strain;the detection of the pagP gene deletion was performed on the membrane by the cell self-polymerization experiment,the outer membrane hydrophobicity test,and the membrane formation condition analysis.The effect of the formation capacity was observed by scanning electron microscopy on the morphology of the pellicle.The results showed that the MIC of the strain decreased after the deletion of pagP gene,and the outer membrane permeability of the strain increased,and the self-polymerization ability of the strain increased significantly(p<0.01).However,the absence of the pagP gene had no significant effect on the hydrophobicity of the outer membrane of the strain.The analysis showed that the bacteria in the LB medium were more stable in the biofilm formation with time.The deletion of pagP gene can affect the expression of virulence,drug resistance and outer membrane related genes in APEC.The deletion of pagP gene can enhance the ability of antiserum complement bactericidal(Serum concentrations of 0.5% and 5%)and anti-phagocytic on APEC;The outer membrane characteristics of APEC changed,and the capacity of pellicle formation was enhanced,indicating that the pagP gene has a negative feedback effect on the virulence of APEC.